The Study Of Sodium Butyrate Intervention In Rat With Focal Cerebral Ischemia Injury Based On Nur77

Focal cerebral ischemia is one of the major diseases that threaten human health fearfully.When the focal cerebral ischemia occurs,the middle cerebral artery and other arteries of the patient will be blocked,hindering the circulation of blood vessels,resulting in hypoxia and lack of energy;and inducing irreversible damage to the brain caused by release of ROS(reactive oxygen species),glutamate toxicity,inflammation,etc.So far,tPA(thrombolytic therapy)is the most effective measure of this type of disease,but the time window for tPA is so narrow that it is not the best ideal.Therefore,it has become the primary problem in current research that improving the therapeutic effect of focal cerebral ischemia and reducing the mortality of patients.Histone deacetylase inhibitor(HDACi),Sodium butyrate(NaBu),is the sodium salt of short-chain fatty acid butyrate.In an animal model of focal cerebral ischemia,Sodium butyrate can reduce the area of cerebral infarction,reduce the size of focal cerebral edema,and inhibit the expression of inflammatory factors such as IL-1? and IL-8.But the mechanism of anti-inflammation has not yet been elucidated.The study found that cerebral ischemia can abnormally activate NLRP3 inflammasome,promote the secretion of biologically active inflammatory factors and induce apoptosis.When NLRP3 inflammasome is activated,it activates the production of the inflammatory factor IL-1?,which induces apoptosis.Clinical studies have reported that NLRP3 protein levels are higher in patients with focal stroke.Previous studies have found that in the rat model of middle cerebral artery occlusion(MCAO),ischemic injury induces NLRP3 inflammasome activation,IL-1? and other inflammatory factors are increased,and inhibition of NLRP3 expression can alleviate the ischemic injury.It suggests that NLRP3 activation is one of the therapeutic targets for focal cerebral ischemic injury.Nur77 protein is a member of the orphan nuclear receptor superfamily.The expression of Nur77 may be involved in the development and progression of cardiovascular diseases such as atherosclerosis by regulating inflammatory factors.In an animal model of atherosclerotic disease,up-regulation of Nur77 expression in microglia can inhibit the expression of pro-inflammatory genes iNOS,IL-1?,TNF-?,and so on.The expression of Nur77 is regulated by histone deacetylase(HDAC)activity,it will promote Nur77 expression by inhibiting the recruitment of HDAC in the Nur77 promoter region.Studies have also shown that Nur77 is involved in the development of focal cerebral ischemic injury,but the regulation mechanism of its expression and the anti-inflammatory mechanism in ischemic injury have not been clarified.In summary,we hypothesized that Sodium butyrate may inhibit the inflammatory response induced by ischemic brain injury by regulating the expression of Nur77 in brain tissue,and the inflammatory component of NLRP3 inflammasome as the main influence of ischemic brain injury,it may be the main target of Nur77.Research objective:The purpose of this study is to clarify that the protective mechanism of sodium butyrate on brain damage caused by focal cerebral ischemia: through in vitro and in vivo experiments,we will discuss HDACi Sodium butyrate alleviate brain injury induced by MCAO based on the activation of Nur77 pathway,inhibition of NLRP3 inflammasome expression.Research method:Part of the experimental animal,we selectted 250g-280 g Wistar male rats and the middle cerebral artery occlusion model was used to simulate focal cerebral ischemia condition.Sodium butyrate(300 mg/kg)was continuously administered 7 days before surgery by means of prophylactic administration.The samples were sacrificed 24 hours after surgery.The TTC staining was used to measure the area of cerebral infarction;Longa grade point standard was used to measure the neurological scores of rats;HE staining was used to observed the morphology of the rat brain tissue,immunohistochemistry was used to observed the level of macrophage in rat brain tissue.We use Western blot and qPCR to measure the change of NLRP3.Then we give the rats Csn-B(15mg/kg,30mg/kg)by postoperative administration for 30 min,and rats were sacrificed at 24 hours after operation.Western blot was used to detect the effect of Nur77 on NLRP3 protein.We measure the expression of Nur77,NLRP3,IL-1?,Caspase1,ERK,p-ERK,CREB,p-CREB proteins in rat brain tissue by Western blot;measure the level of Nur77,NLRP3,IL-1? and Caspase1 mRNA by qPCR.In the experimental part of the cell,the HT22 cell line was used to establish the oxygen and glucose deprivation(OGD)model.MTT was used to explore the optimal concentration of NaBu.Then the experimental cells were grouped to CON,OGD,OGD+NaBu 0.5mM,OGD+NaBu 1mM.We measure the expression of Nur77,NLRP3,IL-1?,Caspase1,ERK,p-ERK,CREB,p-CREB proteins in cell by Western blot;measure the fluorescence intensity of Nur77 and NLRP3 by immunofluorescence technique.Finally,the cells were given Sodium butyrate and Csn-B,and the protein expression of Nur77 and NLRP3 was detected by Western blot.Research results:(1)The MCAO model was established after Sodium butyrate was administered to rats.The TTC staining results show that Sodium butyrate can reduce the area of cerebral infarction;the Longa grade point show that Sodium butyrate can decrease the neurological score;the HE staining and immunohistochemistry results show that Sodium butyrate can improve the morphology of brain tissue neurons,reduce the nerve tissue shrinkage and nuclear karyopyknosis.(2)Establishment of MCAO model.The Western blot results shows that,compared with the CON group,the expression level of NLRP3 protein increased;Different concentrations of Nur77 agonist Csn-B were administered to MCAO rats,which reduced the expression level of NLRP3 in a dose-dependent manner,and high concentration of Csn-B significantly decreased the expression of NLRP3.(3)The MCAO model was established after Sodium butyrate was administered to rats.The Western blot and qPCR results show that Sodium butyrate could improve the expression of Nur77 and p-ERK protein decreased by cerebral ischemia,the expression of NLRP3,IL-1? and Caspase1 increased by cerebral ischemia;ERK,p-CREB,CREB protein has no markedly change.(4)The OGD model of HT22 cells was established.The MTT results show that the optimal concentration of Sodium butyrate is 1mM.The results of Western blot show that Sodium butyrate can improve the expression of Nur77 and p-ERK protein induced by OGD,and weaken the NLRP3,Caspase1 and IL-1? protein expression increased by OGD;ERK,p-CREB,CREB protein has no markedly change.The results of immunofluorescence show that Sodium butyrate can improve the fluorescence intensity of Nur77 induced by OGD,weaken the fluorescence intensity NLRP3 increased by OGD.The Western blot result shows Sodium butyrate can enhance the ability of Nur77 agonist to regulate NLRP3.Research conclusion:(1)Sodium butyrate can reduce the infarct size of rats with focal cerebral ischemia,reduce the neurological score,protect the morphology of neurons,and improve the survival rate of HT22 cells under oxygen-glucose deprivation conditions.(2)Sodium butyrate inhibits the expression of NLRP3 by up-regulating the expression of Nur77 protein and protects focal cerebral ischemic injury in rats.